Drosophila melanogaster 5S ribosomal RNA is transcribed as a precursor, processed by removal of 15 nt form its 3' end and incorporated into the large ribosomal subunit. In vitro 5S RNA processing using an S100 extract form Drosophila melanogaster culture cells is prevented by EDTA, proteinase K digestion or heating to 37 degrees C, suggesting dependence on divalent cations and proteins. Preincubation of extract with RNases followed by the proper RNase inhibitors does not affect processing, indicating that endogenous S100 RNA is probably not directly required for Drosophila melanogaster 5S RNA processing. 135 nt substrate 5S RNA primary transcript was obtained from a T7 RNA polymerase reaction using a Drosophila melanogaster 5S RNA gene linked to a T7 promoter. This construction allowed the investigator to delete domains of mature 5S RNA and test these mutant RNAs for their ability to be processed. Mature 5S RNA consists of five conserved stem-loop domains. All of stem IV and half of stem V are dispensable for processing, while processing identity elements are widely dispersed throughout the rest of 5S RNA. This proposal concerns the fractionation of the S100 extract to investigate processing and 5S RNA binding components. Proteins from the extract can be separated by ion exchange and affinity chromatography. Processing reactions will be performed by incubating mixtures of column fractions with 5S RNA primary transcript and analyzed by denaturing polyacrylamide gel electrophoresis. Faithful 3' end formation has been investigated by preparative processing, two dimensional RNase T1 fingerprinting and secondary nuclease analysis. Binding experiments with column fractions will enable the investigator to correlate specificity of 5S RNA-protein interactions with processing activity. These experiments include nondenaturing polyacrylamide gel shifts, footprinting with single endlabeled 5S RNA, and photoaffinity crosslinking with radioactively labeled RNA to visualize the binding proteins. The proposed research dissects a functionally important and RNA structurally complex RNA-protein interaction.